The covalent chemical attachment of an active moiety to a carrier is a fundamental process that lies at the heart of a diverse range of scientific disciplines. For example, the attachment of peptides, oligosaccharides, DNA or small bioactive organic molecules to glass slides or chips has given rise to the enormous field of diagnostic screening, with example applications such as toxicological testing of new chemical entities and genetic products. Attachment of the same type of molecules to polymeric surfaces has applications in diverse fields such as affinity purification of small molecules/proteins and smart wound dressings that elicit a physiological response to enhance the wound healing process. Alternatively, attachment of such molecules to immunostimulatory proteins has application in the field of synthetic vaccine development. Within each of these fields, successful application is dependent upon the stringent control of a number of key chemical and physiochemical parameters being achieved.
A key objective is to obtain quantitative and qualitative control of the covalent attachment chemistry since this should provide a final construct that exhibits an optimal combination of molecular display and physiochemical characteristics. Each application has many subtle variations of these key requirements to consider, given the range of chemical diversity and intrinsic characteristics present in active moieties such as peptides, oligosaccharides, DNA or small bioactive organic molecules and the different physiochemical properties of a glass slide when compared to a polymeric bead or a protein.
International patent application publication no. WO 01/71043 discloses bonding of peptides and biological molecules to semiconductor nanocrystals using chemical coupling methods. Polynucleotides along with polypeptides and other polymeric biologicals are bound to semiconductor nanocrystals and dicarboxylic acids may be used as surface ligand linker molecules. This method appears to employ a functional group in the linker molecule (e.g. the dicarboxylic acid) for reaction with the peptide to achieve bonding. The semiconductor nanocrystals are typically bonded to a substrate or microsphere.
WO 03/087824, discloses coupling reactions using H-benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP) in Example 1B. WO 06/060664, discloses coupling reactions using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) in Examples 1-2.
U.S. Pat. No. 6,326,144 discloses a reaction for coupling proteins and peptides to water soluble quantum dots. Specifically, Example 3 in column 19 couples avidin (a protein) to water soluble quantum dots using (1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride) (EDAC) and N-hydroxysuccinimide (NHS).
U.S. patent application publication no. US 2006/0068506 A1 discloses a method for bonding a DNA biomolecule to a quantum dot in FIG. 9 and paragraph [0021]. In this method, dihydrolipoic or dihydrolipoic acid/polyethylene glycol-modified core shell quantum dots are bound to a DNA biomolecule using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) as coupling agents.
Methods for preparing water-soluble quantum dots involve the capping of core-shell nanocrystals with ionized dihydrolipoic acid molecules and more recently the use of poly(ethylene glycol) terminated dihydrolipoic acid (DHLA-PEG) ligands with PEGs of various sizes. Electrostatic self-assembly and the metal ion affinity of histidine coordinate techniques may be used to prepare bioconjugates. Electrostatic self-assembly takes advantage of positively charged domains of proteins coupled to negatively charged carboxylate groups on the quantum dot surface (Mattoussi, H., et al., “Self-assembly of CdSe—ZnS quantum dot bioconjugates using an engineered recombinant protein,” J. Am. Chem. Soc., 122, 12142-12140 (2000); Goldman, E. R., et al., “Conjugation of luminescent quantum dots with antibodies using an engineered adaptor protein to provide new reagents for fluoroimmunoassays,” Anal. Chem., 74, 841-847 (2002); and Medintz, I. L., et al., “Self-assembled nanoscale biosensors based on quantum dot FRET donors,” Nature Materials, 2, 630-638 (2003)). Limitations of using electrostatic self-assembly combined with DHLA-capping are that activities must be carried out in a basic environment (pH>7) and there is an inability to form direct covalently linked nanocrystal-biomolecule conjugates in aqueous environments with N-3-(dimethylamino)-propyl-N′-ethyl-carbodiimide (EDC). Employing DHLA-PEG ligands permitted expansion of the range of pH solubility while still permitting electrostatic self-assembly (Uyeda, H. T., et al., “Design of water-soluble quantum dots with novel surface ligands for biological applications,” Mater. Res. Soc. Symp. Proc., 789, 111-116 (2004) and H. T. Uyeda et al., “Design of water-soluble quantum dots with novel surface ligands for biological applications,” J. Am. Chem. Soc. 127, 3870-3878 (2005)).
Small organic surface ligands such as mercaptoacetic acid (MAA), aminoethane thiol (AET) and polymer encapsulation have been used to generate water-soluble quantum dot systems (Chan, W. C. W., et al., “Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic Detection,” Science 281, 2016-2018 (1998)). Water solubilization of quantum dots with hydrophilic dendritic structures and layer-by-layer assembly techniques have also been demonstrated with some degree of success. Such strategies, however, do not provide for pH stability and in some cases do not provide long-term water solubility. The major disadvantage of these systems is the poor temporal stability of the quantum dot-ligands due to the dynamic nature of the singly bound thiol groups, which results in aggregated solutions after a short time. Another disadvantage is the lack of the provision of compact hydrophilic quantum dots and quantum dot bioconjugates that can be easily delivered inside live cells and regions of cells and assay studies such as those based on fluorescence or Foster resonance energy transfer (FRET).
There remains a need in the art for improved coupling methods for covalent attachment of peptides and biological molecules to, for example, luminescent semiconductor nanocrystals or other nanoparticles.